Beyond genetics: What the new fields of functional genomics and epigenetics are revealing about autism

﻿﻿Today’s guest post comes to us from Valerie W. Hu, Ph.D. Dr. Valerie Hu is a Professor of Biochemistry and Molecular Biology at The George Washington University Medical Center as well as a mother of a son with ASD. She redirected her research focus towards autism about 5 years ago and has since published 6 papers on the genes and biological pathways associated with ASD. Dr. Hu received her Ph.D. in Chemistry from Caltech and did her postdoctoral research in Membrane Biochemistry and Immunology at UCLA. More information about her research and papers can be obtained at: http://www.gwumc.edu/biochem/faculty_vhu.html

For many years, genetics has followed the traditional approach to identifying genes associated with various disorders, including autism. However, the wide diversity of symptoms and behaviors associated with autism spectrum disorders (ASD) has posed a significant challenge to identifying mutations in one or a few genes that are reliably associated with ASD. More recent attention has focused on copy number variants (CNVs) which are submicroscopic regions of chromosomes that have been found to be lost or duplicated in some individuals with ASD. Together, these studies are making progress in identifying a number of genes that function at the synapse between nerve cells. Nevertheless, the combination of all known genetic mutations still does not account for the majority of autism cases or for the association of non-neurological symptoms observed in autistic individuals.

My laboratory at The George Washington University Medical Center has taken a functional genomics approach to studying genes that may be deregulated in autism. Rather than identify mutations in DNA, our goal has been to identify genes whose activity, as indicated by gene expression level, is altered in order to identify dysfunctional biochemical pathways and impaired cellular functions in autism. Thus, using a method known as DNA microarray analysis to profile gene expression in cells from identical twin pairs who are differentially diagnosed with autism (one autistic, the other not reaching the threshold for an autism diagnosis), sibling pairs where only one sibling is autistic, and unrelated case-controls, we identified many genes whose expression levels (activities) are different from non-autistic individuals. Furthermore, by subtyping the unrelated autistic individuals according to symptomatic severity across over 63 symptoms probed by a commonly used diagnostic assessment instrument (Autism Diagnostic Interview-Revised), we were able to identify gene “signatures” for each of 3 ASD subtypes studied. These signatures not only revealed genes unique to a given subtype of autism, but also overlapping genes that presumably control common symptoms of autism across subtypes. Interestingly, a set of genes that regulate the biological clock (that is, circadian rhythm) were found to be disrupted only in the subtype of ASD exhibiting severe language impairment.

These studies demonstrating different gene expression signatures between identical but differentially diagnosed twins as well as revealing differential expression of hundreds to thousands genes depending on ASD subtype suggest the involvement of “master switches” (or epigenetic mechanisms) in the control of gene expression. Two new studies by our laboratory, published in two different journals on Apr. 7, 2010, suggest that epigenetics may play a significant role in the regulation of gene expression in autism (read a blog from AS staff on epigenetics). In one study, published in the FASEB Journal, we identified chemical tags (called “methyl groups”) on the DNA of individuals with autism that led to gene silencing. This mode of turning off a gene is potentially reversible with the proper drugs if the specific gene can be properly targeted. The second study, which was published in the journal Genome Medicine, reports on the differential expression of microRNA in autism. MicroRNA are recently discovered snippets of RNA (ribonucleic acid), each of which can inhibit the expression (and thus activity) of hundreds of genes. The effects of microRNA are also reversible by treatment with complementary “anti-sense” RNA. While methylation inhibits gene expression at the level of DNA, microRNA inhibits at the level of RNA. These two studies together illustratetwo different “epigenetic” mechanisms controlling gene activity in autismthat lie beyond genetic mutations.

In the study published in the FASEB Journal, we again used cell lines derived from identical twins and sibling pairs in which only one of the twins or siblings was diagnosed with autism to identify chemical changes on DNA. We then compared the genes that showed changes in DNA tagging (methylation) with a list of genes that showed different levels of expression from these same individuals. The amounts of protein produced by two of the genes that appear on both lists were then investigated in brain tissues from the cerebellum and frontal cortex of autistic and control subjects which were obtained through the Autism Tissue Program. We found that both selected proteins, RORA (retinoic acid-related orphan receptor-alpha) and BCL-2, as predicted by the observed increase in methylation, were reduced in the autistic brain. Although BCL-2 has previously been reported to be reduced in autistic brain, RORA is a novel gene which is relevant to many of the observed deficits in autism. Specifically, RORA is involved in several key processes negatively affect by autism, including Purkinje cell differentiation, cerebellar development, protection of neurons against oxidative stress, suppression of inflammation, and regulation of circadian rhythm.

These results suggest that blocking the chemical tagging of these genes may reverse some symptoms of autism if targeted removal of methyl groups from specific genes can be accomplished. Furthermore, this study, which links molecular alterations in blood-derived cells to brain pathobiology, demonstrates the feasibility of using more easily accessible cells from blood (or other non-brain tissues) for diagnostic screening.

This research is reported in the study, titled “Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain,” which was recently published in the Federation of American Societies for Experimental Biology (FASEB) Journal, and is available online at: http://www.fasebj.org.

In the study published in Genome Medicine, we identified changes in the profile of microRNAs between twins and sibling pairs, again discordant for diagnosis of autism. We discovered that, despite using cells derived originally from blood, brain-specific and brain-related microRNAs were found to be differentially expressed in the autistic samples, and that these microRNAs could potentially regulate genes that control many processes known to be disrupted in autism. For example, differentially expressed miRNAs were found to target genes highly involved in neurological functions and disorders in addition to genes involved in gastrointestinal diseases, circadian rhythm signaling, and steroid hormone metabolism. The study further shows that by treating the cells with antisense RNA antagonists (inhibitors) to specific microRNA or by employing mimics of a particular microRNA, one can reverse the pattern of expression of a given target gene regulated by that microRNA.

This study, titled “Investigation of post-transcriptional gene regulatory networks associated with autism spectrum disorders by microRNA expression profiling of lymphoblastoid cell lines” was highlighted as an “Editor’s pick” in the journal Genome Medicine. It is available online at: http://genomemedicine.com/content/2/4/23.

By integrating both DNA methylation and miRNA expression studies with gene expression data, Dr. Hu and colleagues are applying a systems biology approach to understanding this complex disorder.

Beyond genetics by Dr. Valerie Hu is very interesting.
I have a son who appeared to be the typical “all boy”, until age four. It was then, that he became what was diagnosed as a mute and later a selective mute. Many pediatricians, psychiatrists, psychologists, behavior specialists,family therapists and one hospitalization gave us nothing. He received a negative reputation in elementary school, began acting out to the point where I had to have space. He went to live with his dad, step-mom and their children. He was back and forth between homes, for almost four years. He went back into the same school system, he was in for elementary school. He was non-compliant. One day, he brought a tiny gun in his backpack. He was expelled, permanently. I was told, I had to find a school. Being a mute, his many evaluations didn’t portray an accurate picture. When he was almost 21, he had another evaluation. He responded verbally, when necessary. Results were aspergers/ autistic tendencies. A little late to change his horrible childhood. Happy to finally have something to work with, though. Since then, my cousin’s daughter was diagnosed with Autism. So far, my daughter is healthy. I apologize for the long e-mail. Guess I still need more healing. There is hope for people with Aspergers and Autism. Thank you.

Thank you very much indeed for sharing this most informative and thought-provoking article. I am most impressed at the range and quality of research being published here, and am delighted to be able to access these cutting-edge developments through the Facebook group.

My eleven year-old son has severe regressive autism and developmental delay. His early accelerated development peaked around eighteen months, whereupon he regressed over the following three years into a profoundly autistic child with no functional speech and a very limited understanding of language and the world around him. As parents, the desire to ease the suffering and confusion our children encounter in their daily lives is always uppermost in our thoughts. It is comforting to have access to current research in an original, unsimplified form, allowing us the opportunity both to be kept informed, and treated with dignity.

This article does indeed seem to sow seeds of hope in the prospect of ameliorative treatments in the future, especially for children with speech-centre disruption. It is encouraging and comforting to know that work like this is ongoing.

Suzanne, I feel for you and your son. May our boys benefit from research such as this in the not too distant future.

My thanks to Dr Hu for her wonderful work for autistics. It has been depressing reading about the thousands of genes involved in autism. I feel better now that a linking mechanism has been identified. My son is not dissimilar to Susan’s son although much older.
Are there any indications on what the time frames are for any therapeutic intervention to become available?

This article sparked and interest. I have identical 9 year old twin nephews. One is autistic and the other not. My family has tried every medical and alternative method of treatment. I was wondering if there might be available an identical twin geneology study that can help our situation.

extremely informative and interesting…and it fits in perfectly with other research I’ve read. Very hopeful for what will be done in the future…thanks to all the hard-working REAL scientists out there (so sick of hearing about the pseudo-science out there!).